Ovarian Cancer Methylome

ABSTRACT

The present invention provides methods and kits for identifying a cell that exhibits or is predisposed to exhibiting unregulated growth by detecting hypennethylation of a gene or a regulatory region in at least one gene in the cell. Also provided are methods for diagnosis or prognosis of ovarian cancer in a subject. Also provided are methods of ameliorating ovarian cancer in a subject by administering to the subject an agent that demethylates a hypermethylated gene or regulatory region thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of 13/203,480 filed Sep. 28, 2011 whichis a 35 USC §371 National Stage application of International ApplicationNo. PCT/US2010/025661 filed Feb. 26, 2010, now pending; which claims thebenefit under 35 USC §119(e) to U.S. Application Ser. No. 61/179,777filed May 20, 2009 and to U.S. Application Ser. No. 61/156,218 filedFeb. 27, 2009, both now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application

FIELD OF THE INVENTION

The invention relates generally to methods and kits useful fordetecting, diagnosing or evaluating cancer and more specifically tomethods and kits for detecting, diagnosing or evaluating ovarian cancerby detecting methylation changes in nucleic acid samples of subjectswith a profile of gene markers.

BACKGROUND INFORMATION

Ovarian cancer is the second most common gynecological cancer and theleading cause of death among gynecological cancers worldwide.Approximately 21,650 new cases were detected in 2008 in the UnitedStates, leading to approximately 15,520 deaths from this cancer. Seventypercent of patients with ovarian cancer have advanced disease (stage IIor IV) at presentation, with a 5-year survival rate of 15 to 20% despiteaggressive treatment, while patients presenting with early disease havea survival rate above 90%. The high mortality of ovarian cancer isrelated to the absence of symptoms in the majority of the cases duringthe early stages of the disease, and also to the lack of truly sensitiveand specific screening techniques. The best studied serum biomarker forovarian cancer is CA-125, which is elevated in approximately 80% ofwomen with advanced disease, but only 50-60% in patients withearly-stage disease.

It has been shown that genetic and epigenetic changes contribute to thedevelopment and progression of tumor cells. Epigenetic alterations inpromoter methylation and histone acetylation have been associated withcancer-specific expression differences in human malignancies.Methylation has been primarily considered as a mechanism of tumorsuppressor gene (TSG) inactivation, and comprehensive whole-genomeprofiling approaches to promoter hypermethylation have identifiedmultiple novel putative TSGs silenced by promoter hypermethylation.

Understanding the epigenetic changes that lead to cancer progressionwill help unravel key biologic processes that lead to cancer formation.Thus, there is an imperative need to find new molecular markers thatwill: a) help determine the risk of developing cancer to considerappropriate preventive interventions; b) help detect cancers early whenthey are amenable to surgical cure; c) help to predict response of aparticular therapy (such as paclitaxel); and d) help to determine theoverall outcome of a cancer patient.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that some genes havepromoters that are hypermethylated in cancer. As a result, typically thegene expression is down-regulated. This discovery is useful for cancerscreening, risk-assessment, prognosis, and identification of subjectsresponsive to a therapeutic regimen. Accordingly, there are providedmethods for detecting a cellular proliferative disorder (e.g., ovariancancer) in a subject. The methods of the invention are useful fordiagnostic, prognostic as well therapeutic prediction.

In one aspect, the invention provides a method for diagnosing ovariancancer in a subject having or at risk of developing ovarian cancer. Themethod includes determining the methylation state of a gene or aregulatory region of a gene in at least two genes. By way of example,such genes may include at least one gene selected from the groupconsisting of GULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM,MGMT, KIFIA, CCNA1, ESR1, SSBP2, GSTP1, FKBP4, VGF, and any combinationthereof, in a nucleic acid sample from the subject. A hypermethylatedstate, as compared to a corresponding normal cell, is indicative of asubject having or at risk of developing ovarian cancer. In oneembodiment, at least two genes or regulatory regions are hypermethylatedand the at least two genes are coordinately silenced in the cellundergoing unregulated cell growth. In another embodiment, the methodincludes contacting a nucleic acid-containing sample from cells of thesubject with an agent that provides a determination of the methylationstate of at least one gene or at least one regulatory region of a gene,wherein the at least one regulatory region is hypermethylated in a cellundergoing unregulated cell growth as compared to a corresponding normalcell; and identifying hypermethylation of the regulatory region in thenucleic acid-containing sample, as compared to the same region of the atleast one regulatory region in a subject not having ovarian cancer,wherein hypermethylation is indicative of a subject having or at risk ofdeveloping ovarian cancer.

In another aspect, the invention provides a method for diagnosing cancerin a subject having or at risk of developing a cell proliferativedisorder. The method includes determining the methylation state of atleast one gene or a regulatory region of the at least one gene. By wayof example, such genes may include at least one gene selected from thegroup consisting of GULP1 and CSGALNACT2, in a nucleic acid sample fromthe subject. A hypermethylated state, as compared to a correspondingnormal cell, is indicative of a subject having or at risk of developinga cell proliferative disorder. In one embodiment, at least two genes orregulatory regions are hypermethylated and the at least two genes arecoordinately silenced in the cell undergoing unregulated cell growth. Inanother embodiment, the method includes contacting a nucleicacid-containing sample from cells of the subject with an agent thatprovides a determination of the methylation state of at least one geneor at least one regulatory region of a gene, wherein the at least oneregulatory region is hypermethylated in a cell undergoing unregulatedcell growth as compared to a corresponding normal cell; and identifyinghypermethylation of the regulatory region in the nucleic acid-containingsample, as compared to the same region of the at least one regulatoryregion in a subject not having ovarian cancer, wherein hypermethylationis indicative of a subject having or at risk of developing a cellproliferative disorder.

In another aspect, the invention provides a method of determining theprognosis of a subject having ovarian cancer. The method includesdetermining the methylation state of a gene or a regulatory region of agene in at least two genes. By way of example, such genes may include atleast one gene selected from the group consisting of GULP1, CSGALNACT2,PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA, CCNA1, ESRI, SSBP2,GSTP1, FKBP4, VGF, and any combination thereof, in a nucleic acid samplefrom the subject. A hypermethylated state, as compared to acorresponding normal cell in the subject or a subject not having thedisorder, is indicative of a poor prognosis. In one embodiment, at leasttwo genes or regulatory regions are hypermethylated and the at least twogenes are coordinately silenced in the cell undergoing unregulated cellgrowth.

In another aspect, the invention provides a method of determining theprognosis of a subject having cancer. The method includes determiningthe methylation state of at least one gene or a regulatory regionthereof. By way of example, such genes may include at least one geneselected from the group consisting of GULP1 and CSGALNACT2, in a nucleicacid sample from the subject. A hypermethylated state, as compared to acorresponding normal cell in the subject or a subject not having thedisorder, is indicative of a poor prognosis. In one embodiment, at leasttwo genes or regulatory regions are hypermethylated and the at least twogenes are coordinately silenced in the cell undergoing unregulated cellgrowth.

In another aspect, the invention provides a method of amelioratingsymptoms associated with ovarian cancer in a subject in need thereof.The method includes administering to the subject an agent thatdemethylates at least one gene or regulatory region of a gene. By way ofexample, such genes may include at least one gene selected from thegroup consisting of GULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3,MCAM, MGMT, KIFIA, CCNA1, ESR1, SSBP2, GSTP1, FKBP4, VGF, and anycombination thereof. Demethylation of the at least one gene orregulatory region of a gene that is in a hypermethylated state, ascompared to that of a subject not having ovarian cancer, increasesexpression of the at least one gene or regulatory region, therebyameliorating the symptoms associated with ovarian cancer. In oneembodiment, at least two genes or regulatory regions are hypermethylatedand the at least two genes are coordinately silenced in the cellundergoing unregulated cell growth.

In another aspect, the invention provides a method of ameliorating acell proliferative disorder in a subject in need thereof. The methodincludes administering to the subject an agent that demethylates atleast one gene or regulatory region of a gene. By way of example, suchgenes may include at least one gene selected from the group consistingof GULP 1 and CSGALNACT2. Demethylation of the at least one gene orregulatory region of a gene that is in a hypermethylated state, ascompared to that of a subject not having ovarian cancer, increasesexpression of the at least one gene or regulatory region, therebyameliorating the symptoms associated with ovarian cancer. In oneembodiment, at least two genes or regulatory regions are hypermethylatedand the at least two genes are coordinately silenced in the cellundergoing unregulated cell growth.

In another aspect, the invention provides a method for determiningwhether a subject is responsive to a particular therapeutic regimen. Themethod includes determining the methylation state of a gene or aregulatory region of a gene in at least two genes. In anotherembodiment, the method includes determining the methylation state of atleast one gene or a regulatory region thereof. By way of example, suchgenes may include at least one gene selected from the group consistingof GULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA,CCNA1, ESR1, SSBP2, GSTP1, FKBP4, VGF, and any combination thereof. Inanother embodiment, the genes include at least one gene selected fromthe group consisting of GULP1 and CSGALNACT2. A hypermethylated state ofthe one or more genes or regulatory regions thereof, as compared withthat of a normal subject, is indicative of a subject who is responsiveto the therapeutic regimen. In one embodiment, the therapeutic regimenis administration of one or more chemotherapeutic agents alone or incombination with one or more demethylating agents such as, but notlimited to, 5-azacytidine, 5-aza-2-deoxycytidine and zebularine. Inanother embodiment, the therapeutic regimen is administration ofcisplatin in combination with paclitaxel.

In another aspect, the invention provides a method for identifying acell that exhibits or is predisposed to exhibiting unregulated growth.The method includes detecting hypennethylation of a gene or regulatoryregion of a gene in at least two genes. By way of example, such genesmay include at least one gene selected from the group consisting ofGULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA,CCNA1, ESR1, SSBP2, GSTP1, FKBP4, VGF, and any combination thereof. Ahypermethylated state of the one or more genes or regulatory regionsthereof, as compared to a corresponding normal cell not exhibitingunregulated growth, identifies the cell as exhibiting or predisposed toexhibiting unregulated growth. In one embodiment, at least two genes orregulatory regions are hypermethylated and the at least two genes arecoordinately silenced in the cell undergoing unregulated cell growth. Inanother embodiment, the cell exhibiting, or predisposed to exhibitingunregulated growth is a cancer cell, such as an ovarian cancer cell.

In another aspect, the invention provides a method for identifying acell that exhibits or is predisposed to exhibiting unregulated growth.The method includes detecting hypermethylation of a gene or a regulatoryregion thereof. By way of example, such genes may include at least onegene selected from the group consisting of GULP1 and CSGALNACT2. Ahypermethylated state of the one or more genes or regulatory regionsthereof, as compared to a corresponding normal cell not exhibitingunregulated growth, identifies the cell as exhibiting or predisposed toexhibiting unregulated growth. In one embodiment, at least two genes orregulatory regions are hypermethylated and the at least two genes arecoordinately silenced in the cell undergoing unregulated cell growth. Inanother embodiment, the cell exhibiting, or predisposed to exhibitingunregulated growth is a cancer cell, such as an ovarian cancer cell, atesticular cancer cell, or a bladder cancer cell.

In another aspect, the invention provides a kit useful for the detectionof a methylated CpG-containing nucleic acid in determining themethylation status of one or more genes or regulatory regions thereof.By way of example, such genes may include at least one gene selectedfrom the group consisting of GULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC,PAK3, MCAM, MGMT, KIFIA, CCNA1. ESR1, SSBP2, GSTP1, FKBP4, VGF, and anycombination thereof. In one embodiment, the kit includes a carrierelement containing one or more containers comprising a first containercontaining a reagent that modifies unmethylated cytosine and a secondcontainer containing primers for amplification of the one or more genesor regulatory regions thereof, wherein the primers distinguish betweenmodified methylated and unmethylated nucleic acid. In anotherembodiment, the kit further includes a panel of two or more genesselected from the group consisting of GULP1, CSGALNACT2, PGP9.5, HIC1,AIM1, APC, PAK3, MCAM, MGMT, KIFIA, CCNA1, ESR1, SSBP2, GSTP1, FKBP4,VGF, and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial diagram showing a gene selection flow chart fordiscovery of genes regulated by promoter methylation in ovarian cancer(Discovery Approach).

FIGS. 2A and 2B are pictorial diagrams showing another gene selectionflow chart for testing of genes regulated by promoter methylation inovarian cancer (Candidate Approach). FIG. 2A shows the Samples set forCandidate Approach Analyses. FIG. 2B shows a flowchart of Analyses withQuantitative Methylation Specific PCR (QMSP).

FIG. 3 is a graphical diagram showing promoter methylation levels forthe CSGALNACT2 gene in the ovarian cancer patient samples (Tumors; n=57)and normal ovary tissues (Controls; n=15). The quantity of eachmethylated gene promoter was expressed as the ratio of the amount of PCRproducts amplified from the methylated gene to the amount amplified withthe reference gene β-actin multiplied by 1,000.

FIG. 4 is a pictorial diagram showing results of a conventionalmethylation specific PCR. PCR products were electrophoresed on a 8%polyacrilamide gel for the detection of promoter methylation in theGULP1 gene in the ovarian cancer patient samples (Tumors) and normalovary tissues (Controls). The amplicon size is 169 bp. NTC, no templatecontrol; +CTRL, positive control (100% in vitro methylated DNA).

FIG. 5 is a graphical diagram showing promoter methylation levels forthe VGF gene in the ovarian cancer patient samples (Tumors; n=368,borderline tumors; n=15), benign tumors (cystadenoma; n=18) and normalovary tissues (Controls; n=16). The quantity of each methylated genepromoter was expressed as the ratio of the amount of PCR productsamplified from the methylated gene to the amount amplified with thereference gene β-actin multiplied by 1,000.

FIG. 6 is a graphical diagram showing promoter methylation levels forthe PGP9.5 gene in the ovarian cancer patient samples (Tumors; n=373,borderline tumors; n=17), benign tumors (cystadenoma; n=19) and normalovarian tissues (Controls; n=13). The quantity of each methylated genepromoter was expressed as the ratio of the amount of PCR productsamplified from the methylated gene to the amount amplified with thereference gene β-actin multiplied by 1,000.

FIG. 7 is a graphical diagram showing promoter methylation levels forthe PGP9.5 gene in the ovarian cancer patient samples divided in 2groups the ones that responded to chemotherapy treatment (CR+PR; n=144)and the ones that did not respond (SD+PD, n=55). The quantity of eachmethylated gene promoter was expressed as the ratio of the amount of PCRproducts amplified from the methylated gene to the amount amplified withthe reference gene β-actin multiplied by 1,000.

FIG. 8 is a graphical diagram showing promoter methylation levels forthe PGP9.5 gene in the ovarian cancer patient samples subdivided in 4groups the ones that responded to chemotherapy treatment in completeresponse (CR; n=68) and partial response (PR; n=76) and the ones thatdid not respond: Stable disease (SD; n=17), and progressive disease(PD;n=38). The quantity of each methylated gene promoter was expressed asthe ratio of the amount of PCR products amplified from the methylatedgene to the amount amplified with the reference gene β-actin multipliedby 1,000.

FIG. 9 is a graphical diagram showing promoter methylation levels forthe PGP9.5 gene in the ovarian cancer patient samples divided by stage(Early Stage; n=98 and Late Stage; n=273). The quantity of eachmethylated gene promoter was expressed as the ratio of the amount of PCRproducts amplified from the methylated gene to the amount amplified withthe reference gene β-actin multiplied by 1,000. 61.2% of the early stagepatients showed methylation and 49% of the late stage patients showedmethylation.

FIG. 10 is a graphical diagram showing promoter methylation levels forthe PGP9.5 gene in the ovarian cancer patient samples of differenthistology; Serous (n=245), Mucinous (n=43), Endometrioid (n=39), ClearCell (n=20), Adeno (n=15). The quantity of each methylated gene promoterwas expressed as the ratio of the amount of PCR products amplified fromthe methylated gene to the amount amplified with the reference geneβ-actin multiplied by 1,000.

FIG. 11 is a graphical diagram showing promoter methylation levels forthe CSGALNACT2 gene in the ovarian cancer patient samples (Tumors;n=291, borderline tumors; n=14), benign tumors (cystadenoma; n=17) andnormal ovary tissues (Controls; n=16). The quantity of each methylatedgene promoter was expressed as the ratio of the amount of PCR productsamplified from the methylated gene to the amount amplified with thereference gene β-actin multiplied by 1,000.

FIG. 12 is a graphical diagram showing promoter methylation levels forthe CSGALNACT2 gene in the ovarian cancer patient samples divided bystage (Early Stage; n=107 and Late Stage; n=271). The quantity of eachmethylated gene promoter was expressed as the ratio of the amount of PCRproducts amplified from the methylated gene to the amount amplified withthe reference gene β-actin multiplied by 1,000.

FIG. 13 is a pictorial diagram showing representative sequencing resultsof the GULP1 gene in cancer cell lines (IGROV, A2780, 2008), and normalcell lines (OSE2A, OSE2B, OSE7). Arrows, all guanines present aftersequencing are complementary to methyl cytosines on the opposite DNAstrand. The adenines are complementary to timines, respresenting absenceof methylation.

FIG. 14 is a pictorial diagram showing GULP1 expression in normal celllines (OSE2A, OSE2B, OSE7) and cancer cell lines (IGROV, A2780, 2008).Top=RT-PCR to assess RNA level. Bottom=Western blot to assess proteinlevel.

FIGS. 15A-15C are pictorial diagrams showing promoter methylation ofGULP1. FIG. 15A shows methylation of GULP1 by conventionalmethylation-specific PCR in cancer cell lines (IGROV, A2780, 2008), andnormal cell lines (OSE2A, OSE2B, OSE7); methylated; U, unmethylated;NTC, no template control; +CTRL, positive control (100% in vitromethylated DNA). FIG. 15B shows primary ovarian tumors. FIG. 15C showsnormal ovarian tissues.

FIG. 16 is a pictorial diagram showing GULP1 expression in primaryovarian tumors. RT-PCR to assess RNA level.

FIG. 17 is a graphical diagram showing promoter methylation levels forthe GULP1 gene in the ovarian tumor samples of different histology(Tumors; n=422, borderline tumors; n=16), benign tumors (cystadenoma;n=18) and normal ovary tissues (Controls; n=13). The quantity of eachmethylated gene promoter was expressed as the ratio of the amount of PCRproducts amplified from the methylated gene to the amount amplified withthe reference gene β-actin multiplied by 1,000.

FIG. 18 is a graphical diagram showing promoter methylation levels forthe GULP1 gene in the ovarian cancer patient samples divided into twogroups, the ones that responded to chemotherapy treatment (n=158) andthe ones that did not respond (n=67). The quantity of each methylatedgene promoter was expressed as the ratio of the amount of PCR productsamplified from the methylated gene to the amount amplified with thereference gene β-actin multiplied by 1,000.

FIG. 19 is a graphical diagram showing promoter methylation levels forthe GULP1 gene in the ovarian cancer patient samples stratified by stage(Early Stage; n=106 and Late Stage; n=313). The quantity of eachmethylated gene promoter was expressed as the ratio of the amount of PCRproducts amplified from the methylated gene to the amount amplified withthe reference gene β-actin multiplied by 1,000.

FIG. 20 is a graphical diagram showing promoter methylation levels forthe GULP1 gene in the ovarian cancer patient samples divided by before(n=14) and after (n=14) treatment (paired samples are indicated by theconnecting lines). The quantity of each methylated gene promoter wasexpressed as the ratio of the amount of PCR products amplified from themethylated gene to the amount amplified with the reference gene β-actinmultiplied by 1,000.

FIG. 21 is a graphical diagram showing promoter methylation levels forthe GULP1 gene in the urines from bladder cancer patients (n=58) andnormal subjects (n=46). The quantity of each methylated gene promoterwas expressed as the ratio of the amount of PCR products amplified fromthe methylated gene to the amount amplified with the reference geneβ-actin multiplied by 1,000.

FIG. 22 is a graphical diagram showing promoter methylation levels forthe GULP1 gene in the testicular cancer patient samples (Seminomas;n=13, Non-seminomas; n=13), and Normal Testicular Tissues (n=17). Thequantity of each methylated gene promoter was expressed as the ratio ofthe amount of PCR products amplified from the methylated gene to theamount amplified with the reference gene β-actin multiplied by 1,000.

FIG. 23 is a graphical diagram showing analysis of cell growth by aWST-8 Cell Counting Kit-8 (CCK-8 assay). The growth condition of theIGROV CP cells (ovarian cancer cells) were measured after 1, 2, 3 and 4days. Goe ICP, GULP1 overexpression vector; Gev ICP, control vector; RICP, only the transfection reagent was added; C ICP, mock/control.

FIG. 24 is a pictorial diagram showing that ectopic expression of GULP1inhibits tumor cell growth. The effect of ectopic expression of GULP1 oncarcinoma cell clonogenicity was investigated by colony formation assay.Cells were transfected with GULP1 overexpression vector (OE)or controlvector (EV), and selected with G418. HT 1376=Bladder Cancer Cell Lineand 2008 C13=Ovarian Cancer Cell Line.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that several genes havepromoters that are hypermethylated, thus typically resulting intranscriptional down-regulation in cancer. Accordingly, in a firstembodiment of the invention, there are provided methods for identifyinga cell that exhibits or is predisposed to exhibiting unregulated growth.The method includes determining the methylation state of a gene or aregulatory region in at least one gene in the cell, wherein the at leastone gene is hypermethylated as compared to a corresponding normal cellnot exhibiting unregulated growth, thereby identifying the cell asexhibiting or predisposed to exhibiting unregulated growth.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

It has been shown that genetic changes, which include deletions,amplifications, and mutations in DNA sequence, and epigenetic changes,which refer to heritable changes in the gene expression that occurwithout changes to the DNA sequence, contribute to the development andprogression of tumor cells.

The genes or regulatory regions thereof whose methylation status isdetected in the methods provided herein can be any gene or regulatoryregion thereof identified as hypermethylated in a cell exhibitingunregulated growth as compared to a corresponding normal cell, notundergoing unregulated cell growth. In certain embodiments, at least twogenes or regulatory regions are hypermethylated and the at least twogenes are coordinately expressed in the cell undergoing unregulated cellgrowth. In other aspects, at least three, or at least four, or at leastfive, or more genes or regulatory regions are hypermethylated.

As used herein, the term “hypermethylated” refers to the addition of oneor more methyl groups to a cytosine ring in a DNA sequence to formmethyl cytosine as compared to a “normal” gene. Such methylationstypically only occur on cytosines that precede a guanosine in the DNAsequence, which is commonly known as a CpG dinucleotide. There areCpG-rich regions known as CpG islands which span the 5′ end region(e.g., promoter, untranslated region and exon 1) of many genes and areusually unmethylated in normal cells. The methylation patterns of cancercells are altered as compared to the corresponding normal cells,undergoing global DNA hypomethylation as well as hypermethylation of CpGislands. Hypomethylation has been hypothesized to contribute tooncogenesis by transcriptional activation of oncogenes and latenttransposons, or by chromosome instability. Aberrant promoterhypermethylation and histone modification, leading to transcriptionalinactivation and gene silencing, is a common phenomenon in human cancercells and likely one of the earliest events in carcinogenesis. As such,hypermethylation of CpG islands in gene promoter regions is a frequentmechanism of inactivation of tumor suppressor genes.

As used herein “corresponding normal cells” means cells that are fromthe same organ and of the same type as the cells being examined, but areknown to be free from the disorder being diagnosed or treated. In oneaspect, the corresponding normal cells comprise a sample of cellsobtained from a healthy individual. Such corresponding normal cells can,but need not be, from an individual that is age-matched to theindividual providing the cells being examined. In another aspect, thecorresponding normal cells comprise a sample of cells obtained from anotherwise healthy portion of tissue of a subject having ovarian cancer.

Accordingly, the present invention is designed to profile methylationalterations on promoter regions of selected genes in ovarian tumors withthe aim of identifying candidate markers for diagnosis and prognosis ofthe disease, with sensitivity and specificity necessary to identifysubjects with early asymptomatic ovarian cancer, as well as diseasemonitoring, therapeutic prediction and new targets for therapy.

In one embodiment, the gene or regulatory region is two or more genesincluding those listed here and/or additional genes (the “targetgenes”). Exemplary target genes include, but are not limited to, atleast one gene or regulatory region thereof selected from GULP1,CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA, CCNA1,ESR1, SSBP2, GSTP1, FKBP4, VGF, and any combination thereof. In someembodiments, the gene or regulatory region thereof is one or more ofGULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA,CCNA1, ESR1, SSBP2, GSTP1, FKBP4, VGF, and any combination thereof.

As provided herein, hypermethylation may occur in the gene or regulatoryregion thereof. In some embodiments, the hypermethylation occurs withinthe regulatory region of the genes identified herein, in particularembodiments, the hypermethylation is in the promoter sequence of theregulatory region. For example, the hypermethylation may be in a CpGdinucleotide motif of the promoter. As such, in one embodiment, themethylation status of the regulatory regions of one or more of GULP1,CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA, CCNA1,ESR1, SSBP2, GSTP1, FKBP4, and VGF, is determined. It is understood thatthis list is not meant to be limiting but instead, illustrative.

Thus, in one aspect, the invention provides a method for diagnosingovarian cancer in a subject having or at risk of developing ovariancancer. The method includes determining the methylation state of a geneor a regulatory region of a gene in at least two genes wherein at leastone gene is selected from the group consisting of GULP1, CSGALNACT2,PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA, CCNA1, ESR1, SSBP2,GSTP1, FKBP4, VGF, and any combination thereof, in a nucleic acid samplefrom the subject, wherein the methylation state is hypermethylated ascompared to a corresponding normal cell, and wherein hypermethylation isindicative of a subject having or at risk of developing ovarian cancer.In another embodiment, the method includes contacting a nucleicacid-containing sample from cells of the subject with an agent thatprovides a determination of the methylation state of at least one geneor a regulatory region of a gene, wherein the at least one regulatoryregion is hypermethylated in a cell undergoing unregulated cell growthas compared to a corresponding normal cell; and identifyinghypermethylation of the regulatory region in the nucleic acid-containingsample, as compared to the same region of the at least one regulatoryregion in a subject not having the proliferative disorder, whereinhypermethylation is indicative of a subject having or at risk ofdeveloping ovarian cancer.

As used herein, the term “cell proliferative disorder” refers tomalignant as well as non-malignant cell populations which often differfrom the surrounding tissue both morphologically and genotypically. Insome embodiments, the cell proliferative disorder is a cancer. Inparticular embodiments the cancer may be a carcinoma or a sarcoma. Acancer can include, but is not limited to, head cancer, neck cancer,head and neck cancer, lung cancer, breast cancer, prostate cancer,colorectal cancer, esophageal cancer, stomach cancer, leukemia/lymphoma,uterine cancer, skin cancer, endocrine cancer, urinary cancer,pancreatic cancer, gastrointestinal cancer, ovarian cancer, testicularcancer, bladder cancer, cervical cancer, and adenomas. In oneembodiment, the cancer is ovarian cancer.

The nucleic acid-containing sample for use in the invention methods maybe virtually any biological sample that contains nucleic acids from thesubject. The biological sample can be a tissue sample which contains 1to 10,000,000, 1000 to 10,000,000, or 1,000,000 to 10,000,000 somaticcells. However, it is possible to obtain samples that contain smallernumbers of cells, even a single cell in embodiments that utilize anamplification protocol such as PCR. The sample need not contain anyintact cells, so long as it contains sufficient material (e.g., proteinor genetic material, such as RNA or DNA) to assess methylation status orgene expression levels.

As used herein, the terms “sample” and “biological sample” refer to anysample suitable for the methods provided by the present invention. Asample of cells used in the present method can be obtained from tissuesamples or bodily fluid from a subject, or tissue obtained by a biopsyprocedure (e.g., a needle biopsy) or a surgical procedure. In oneembodiment, the biological or tissue sample can be drawn from any tissuethat is susceptible to cancer. Thus, exemplary samples include, but arenot limited to, a tissue sample, a frozen tissue sample, a biopsyspecimen, a surgical specimen, a cytological specimen, whole blood, bonemarrow, cerebral spinal fluid, peritoneal fluid, pleural fluid, lymphfluid, serum, mucus, plasma, urine, chyle, stool, sputum, nippleaspirate and saliva. In certain embodiments, the sample can be afraction of a blood sample such as a peripheral blood lymphocyte (PBL)fraction. Methods for isolating PBLs from whole blood are well known inthe art. In addition, it is possible to use a blood sample and enrichthe small amount of circulating cells from a tissue of interest, e.g.,ovaries, breast, etc., using methods known in the art.

The term “subject” as used herein refers to any individual or patient towhich the subject methods are performed. Generally the subject is human,although as will be appreciated by those in the art, the subject may bean animal. Thus other animals, including mammals such as rodents(including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits,farm animals including cows, horses, goats, sheep, pigs, etc., andprimates (including monkeys, chimpanzees, orangutans and gorillas) areincluded within the definition of subject. In addition, the term“subject” may refer to a culture of cells, where the methods of theinvention are performed in vitro to assess, for example, efficacy of atherapeutic agent.

Numerous methods for analyzing methylation status of a gene orregulatory region are known in the art and can be used in the methods ofthe present invention to identify hypermethylation. As illustrated inthe Examples herein, analysis of methylation can be performed bybisulfite genomic sequencing.

Bisulfite ions, for example, sodium bisulfite, convert non-methylatedcytosine residues to bisulfite modified cytosine residues. The bisulfiteion treated gene sequence can be exposed to alkaline conditions, whichconvert bisulfite modified cytosine residues to uracil residues. Sodiumbisulfite reacts readily with the 5,6-double bond of cytosine (butpoorly with methylated cytosine) to form a sulfonated cytosine reactionintermediate that is susceptible to deamination, giving rise to asulfonated uracil. The sulfonate group can be removed by exposure toalkaline conditions, resulting in the formation of uracil. The DNA canbe amplified, for example, by PCR, and sequenced to determine whetherCpG sites are methylated in the DNA of the sample. Uracil is recognizedas a thymine by Taq polymerase and, upon PCR, the resultant productcontains cytosine only at the position where 5-methylcytosine waspresent in the starting template DNA. One can compare the amount ordistribution of uracil residues in the bisulfite ion treated genesequence of the test cell with a similarly treated correspondingnon-methylated gene sequence. A decrease in the amount or distributionof uracil residues in the gene from the test cell indicates methylationof cytosine residues in CpG dinucleotides in the gene of the test cell.The amount or distribution of uracil residues also can be detected bycontacting the bisulfite ion treated target gene sequence, followingexposure to alkaline conditions, with an oligonucleotide thatselectively hybridizes to a nucleotide sequence of the target gene thateither contains uracil residues or that lacks uracil residues, but notboth, and detecting selective hybridization (or the absence thereof) ofthe oligonucleotide.

In another embodiment, the gene is contacted with hydrazine, whichmodifies cytosine residues, but not methylated cytosine residues, thenthe hydrazine treated gene sequence is contacted with a reagent such aspiperidine, which cleaves the nucleic acid molecule at hydrazinemodified cytosine residues, thereby generating a product comprisingfragments. By separating the fragments according to molecular weight,using, for example, an electrophoretic, chromatographic, or massspectrographic method, and comparing the separation pattern with that ofa similarly treated corresponding non-methylated gene sequence, gaps areapparent at positions in the test gene contained methylated cytosineresidues. As such, the presence of gaps is indicative of methylation ofa cytosine residue in the CpG dinucleotide in the target gene of thetest cell.

Modified products can be detected directly, or after a further reactionthat creates products that are easily distinguishable. Means whichdetect altered size and/or charge can be used to detect modifiedproducts, including but not limited to electrophoresis, chromatography,and mass spectrometry. Examples of such chemical reagents for selectivemodification include hydrazine and bisulfate ions. Hydrazine-modifiedDNA can be treated with piperidine to cleave it Bisulfite ion-treatedDNA can be treated with alkali. Other means which are reliant onspecific sequences can be used, including but not limited tohybridization, amplification, sequencing, and ligase chain reaction.Combinations of such techniques can be used as is desired.

In another example, methylation status may be assessed using real-timemethylation specific PCR (QMSP). For example, the methylation level ofthe promoter region of one or more of the target genes can be determinedby determining the amplification level of the promoter region of thetarget gene based on amplification-mediated displacement of one or moreprobes whose binding sites are located within the amplicon. In general,real-time quantitative methylation specific PCR is based on thecontinuous monitoring of a progressive fluorogenic PCR by an opticalsystem. Such PCR systems are well-known in the art and usually use twoamplification primers and an additional amplicon-specific, fluorogenichybridization probe that specifically binds to a site within theamplicon. The probe can include one or more fluorescence labeledmoieties. For example, the probe can be labeled with two fluorescentdyes: 1) a 6-carboxy-fluorescein (FAM), located at the 5′-end, whichserves as reporter, and 2) a 6-carboxy-tetramethyl-rhodamine (TAMRA),located at the 3′-end, which serves as a quencher. When amplificationoccurs, the 5′-3′ exonuclease activity of the Taq DNA polymerase cleavesthe reporter from the probe during the extension phase, thus releasingit from the quencher. The resulting increase in fluorescence emission ofthe reporter dye is monitored during the PCR process and represents thenumber of DNA fragments generated.

In other embodiments, hypermethylation can be identified through nucleicacid sequencing after bisulfite treatment to determine whether a uracilor a cytosine is present at a specific location within a gene orregulatory region. If uracil is present after bisulfite treatment, thenthe nucleotide was unmethylated. Hypermethylation is present when thereis a measurable increase in methylation.

In another embodiment, the method for analyzing methylation of thetarget gene can include amplification using a primer pair specific formethylated residues within the target gene. Thus, selectivehybridization or binding of at least one of the primers is dependent onthe methylation state of the target DNA sequence (Herman et al., Proc.Natl. Acad Sci. USA, 93:9821 (1996)). For example, the amplificationreaction can be preceded by bisulfite treatment, and the primers canselectively hybridize to target sequences in a manner that is dependenton bisulfite treatment. As such, one primer can selectively bind to atarget sequence only when one or more bases of the target sequence isaltered by bisulfite treatment, thereby being specific for a methylatedtarget sequence.

Other methods are known in the art for determining methylation status ofa target gene, including, but not limited to, array-based methylationanalysis (see Gitan et al., Genome Res 12:158-64, 2002) and Southernblot analysis.

Methods using an amplification reaction can utilize a real-timedetection amplification procedure. For example, the method can utilizemolecular beacon technology (Tyagi S., et al., Nature Biotechnology, 14:303 (1996)) or TAQMAN™ technology (Holland, P. M., et al., Proc. Natl.Acad. Sci. USA, 88:7276 (1991)).

In addition, methyl light (Trinh, et al. DNA methylation analysis byMethyLight technology, Methods, 25(4):456-62 (2001), incorporated hereinin its entirety by reference), Methyl Heavy (Epigenomics, Berlin,Germany), or SNuPE (single nucleotide primer extension) (See e.g.,Watson, et al., Genet Res. 75(3):269-74 (2000)) can be used in themethods of the present invention related to identifying alteredmethylation of the genes or regulatory regions provided herein.Additionally, methyl light, methyl heavy, and array-based methylationanalysis can be performed, by using bisulfite treated DNA that is thenPCR-amplified, against microarrays of oligonucleotide target sequenceswith the various forms corresponding to unmethylated and methylated DNA.

The degree of methylation in the DNA associated with the gene or genesor regulatory regions thereof, may be measured by fluorescent in situhybridization (FISH) by means of probes that identify and differentiatebetween genomic DNAs, which exhibit different degrees of DNAmethylation. FISH is described in Human chromosomes: principles andtechniques (Editors, Ram S. Verma, Arvind Babu Verma, Ram S.) 2nd ed.,N.Y.: McGraw-Hill, 1995, and de Capoa A., Di Leandro M., Grappelli C.,Menendez F., Poggesi I., Giancotti P., Marotta, M. R., Spano A., RocchiM., Archidiacono N., Niveleau A. Computer-assisted analysis ofmethylation status of individual interphase nuclei in human culturedcells. Cytometry. 31:85-92, 1998, which is incorporated herein byreference. In this case, the biological sample will typically be anythat contains sufficient whole cells or nuclei to perform short termculture. Usually, the sample will be a tissue sample that contains 10 to10,000, or, for example, 100 to 10,000, whole somatic cells. However, asindicated above, in one embodiment, the biological sample can be atissue sample which contains 1 to 10,000,000, 1000 to 10,000,000, or1,000,000 to 10,000,000 somatic cells.

In another embodiment, methylation-sensitive restriction endonucleasescan be used to detect methylated CpG dinucleotide motifs. Suchendonucleases may either preferentially cleave methylated recognitionsites relative to non-methylated recognition sites or preferentiallycleave non-methylated relative to methylated recognition sites. Examplesof the former are Acc III, Ban I, BstN I, Msp I, and Xma I. Examples ofthe latter are Acc II, Ava I, BssH II, BstU I, Hpa II, and Not I.Alternatively, chemical reagents can be used that selectively modifyeither the methylated or non-methylated form of CpG dinucleotide motifs.

In some embodiments, hypermethylation of the target gene is detected bydetecting decreased expression of the that gene. Expression of a genecan be assessed using any means known in the art. Typically expressionis assessed and compared in test samples and control samples which maybe normal, non-malignant cells. The test samples may contain cancercells or pre-cancer cells or nucleic acids from the cells. Methodsemploying hybridization to nucleic acid probes can be employed formeasuring specific mRNAs. Such methods include using nucleic acid probearrays (microarray technology), in situ hybridization, and usingNorthern blots. Messenger RNA can also be assessed using amplificationtechniques, such as RT-PCR. Advances in genomic technologies now permitthe simultaneous analysis of thousands of genes, although many are basedon the same concept of specific probe-target hybridization.Sequencing-based methods are an alternative; these methods started withthe use of expressed sequence tags (ESTs), and now include methods basedon short tags, such as serial analysis of gene expression (SAGE) andmassively parallel signature sequencing (MPSS). Differential displaytechniques provide yet another means of analyzing gene expression; thisfamily of techniques is based on random amplification of cDNA fragmentsgenerated by restriction digestion, and bands that differ between twotissues identify cDNAs of interest. Moreover, specific proteins can beassessed using any convenient method including, but not limited to,immunoassays and immuno-cytochemistry. Most such methods will employantibodies that are specific for the particular protein or proteinfragments. The sequences of the mRNA (cDNA) and proteins of the targetgenes of the present invention are known in the art and publiclyavailable.

As used herein, the term “selective hybridization” or “selectivelyhybridize” refers to hybridization under moderately stringent or highlystringent physiological conditions, which can distinguish relatednucleotide sequences from unrelated nucleotide sequences.

As known in the art, in nucleic acid hybridization reactions, theconditions used to achieve a particular level of stringency will vary,depending on the nature of the nucleic acids being hybridized. Forexample, the length, degree of complementarity, nucleotide sequencecomposition (for example, relative GC: AT content), and nucleic acidtype, i.e., whether the oligonucleotide or the target nucleic acidsequence is DNA or RNA, can be considered in selecting hybridizationconditions. An additional consideration is whether one of the nucleicacids is immobilized, for example, on a filter. Methods for selectingappropriate stringency conditions can be determined empirically orestimated using various formulas, and are well known in the art (see,for example, Sambrook et al., supra, 1989).

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, for example, highstringency conditions, or each of the conditions can be used, forexample, for 10 to 15 minutes each, in the order listed above, repeatingany or all of the steps listed.

The term “nucleic acid molecule” is used broadly herein to mean asequence of deoxyribonucleotides or ribonucleotides that are linkedtogether by a phosphodiester bond. As such, the term “nucleic acidmolecule” is meant to include DNA and RNA, which can be single strandedor double stranded, as well as DNA/RNA hybrids. Furthermore, the term“nucleic acid molecule” as used herein includes naturally occurringnucleic acid molecules, which can be isolated from a cell, for example,a particular gene of interest, as well as synthetic molecules, which canbe prepared, for example, by methods of chemical synthesis or byenzymatic methods such as by the polymerase chain reaction (PCR), and,in various embodiments, can contain nucleotide analogs or a backbonebond other than a phosphodiester bond.

The terms “polynucleotide” and “oligonucleotide” also are used herein torefer to nucleic acid molecules. Although no specific distinction fromeach other or from “nucleic acid molecule” is intended by the use ofthese terms, the term “polynucleotide” is used generally in reference toa nucleic acid molecule that encodes a polypeptide, or a peptide portionthereof, whereas the term “oligonucleotide” is used generally inreference to a nucleotide sequence useful as a probe, a PCR primer, anantisense molecule, or the like. Of course, it will be recognized thatan “oligonucleotide” also can encode a peptide. As such, the differentterms are used primarily for convenience of discussion.

A polynucleotide or oligonucleotide comprising naturally occurringnucleotides and phosphodiester bonds can be chemically synthesized orcan be produced using recombinant DNA methods, using an appropriatepolynucleotide as a template. In comparison, a polynucleotide comprisingnucleotide analogs or covalent bonds other than phosphodiester bondsgenerally will be chemically synthesized, although an enzyme such as T7polymerase can incorporate certain types of nucleotide analogs into apolynucleotide and, therefore, can be used to produce such apolynucleotide recombinantly from an appropriate template.

In yet another aspect, the invention provides methods of determining theprognosis of a subject having ovarian cancer. The method includesdetermining the methylation state of a gene or a regulatory region of agene in at least two genes in a nucleic acid sample from the subject. Acomparison of the hypermethylation of the gene or regulatory regionthereof, as compared to that of a corresponding normal cell in thesubject or a subject not having the disorder, is indicative of a poorprognosis.

In another aspect, the invention provides methods of identifying a cellthat exhibits or is predisposed to exhibiting unregulated growth. Themethod includes detecting hypermethylation of a gene or regulatoryregion of a gene in at least two genes wherein at least one gene isselected from the group consisting of GULP1, CSGALNACT2, PGP9.5, HIC1,AIM1, APC, PAK3, MCAM, MGMT, KIFIA, CCNA1, ESR1, SSBP2, GSTP1, FKBP4,VGF, and any combination thereof. Hypermethylation of at least one gene,as compared to a corresponding normal cell not exhibiting unregulatedgrowth, identifies the cell as exhibiting or predisposed to exhibitingunregulated growth.

In another aspect, the invention provides methods of amelioratingsymptoms associated with ovarian cancer in a subject in need thereof.The method includes administering to the subject an agent thatdemethylates at least one gene or regulatory region in a gene that ishypermethylated as compared to that of a subject not having thedisorder, thereby reducing expression of the at least one gene andameliorating the symptoms associated with ovarian cancer. The signs orsymptoms to be monitored will be characteristic of ovarian cancer andwill be well known to the skilled clinician, as will the methods formonitoring the signs and conditions.

As used herein, the terms “administration” or “administering” aredefined to include the act of providing a compound or pharmaceuticalcomposition of the invention to a subject in need of treatment.Exemplary forms of administration include, but are not limited to,topical administration, and injections such as, without limitation,intravitreal, intravenous, intramuscular, intra-arterial, intra-thecal,intra-capsular, intra-orbital, intra-cardiac, intra-dermal,intra-peritoneal, trans-tracheal, sub-cutaneous, sub-cuticular,intra-articulare, sub-capsular, sub-arachnoid, intra-spinal andintra-sternal injection and infusion. The phrases “systemicadministration,” “administered systemically,” “peripheraladministration” and “administered peripherally” as used herein mean theadministration of a compound, drug or other material other than directlyinto the central nervous system, such that it enters the subject'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

The total amount of a compound or composition to be administered inpracticing a method of the invention can be administered to a subject asa single dose, either as a bolus or by infusion over a relatively shortperiod of time, or can be administered using a fractionated treatmentprotocol, in which multiple doses are administered over a prolongedperiod of time. One skilled in the art would know that the amount of thecompound or composition to treat ovarian cancer and/or ameliorate thesymptoms associated therewith in a subject depends on many factorsincluding the age and general health of the subject as well as the routeof administration and the number of treatments to be administered. Inview of these factors, the skilled artisan would adjust the particulardose as necessary. In general, the formulation of the pharmaceuticalcomposition and the routes and frequency of administration aredetermined, initially, using Phase I and Phase II clinical trials.

As used herein, the term “ameliorating” or “treating” means that theclinical signs and/or the symptoms associated with cellularproliferative disorder (e.g., ovarian cancer) are lessened as a resultof the actions performed. The signs or symptoms to be monitored will becharacteristic of the cellular proliferative disorder (e.g., ovariancancer) and will be well known to the skilled clinician, as will themethods for monitoring the signs and conditions. Also included in thedefinition of “ameliorating” or “treating” is the lessening of symptomsassociated with ovarian cancer in subjects not yet diagnosed as havingthe specific cancer. As such, the methods may be used as a means forprophylactic therapy for a subject at risk of having ovarian cancer.

As used herein, the term “demethylating agent” is used to refer to anycompound that can inhibit methylation, resulting in the expression ofthe previously hypermethylated silenced genes. Cytidine analogs such as5-azacytidine (azacitidine) and 5-aza-2-deoxycytidine (decitabine) arethe most commonly used demethylating agents. These compounds work bybinding to the enzymes that catalyze the methylation reaction, DNAmethyltransferases. Thus, in one embodiment, the demethylating agent is5-azacytidine, 5-aza-2-deoxycytidine, or zebularine. In anotherembodiment, the demethylating agent is delivered locally to a tumor siteor systemically by targeted drug delivery.

Agents that demethylate the hypermethylated gene or regulatory region ofthe gene can be contacted with cells in vitro or in vivo for the purposeof restoring normal gene expression to the cell. Efficacy of thetreatment can be assessed by detecting decreased expression ordemethylation of a gene selected from the group consisting of GULP1,CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA, CCNA1,ESR1, SSBP2, GSTP1, FKBP4, and VGF.

Once disease is established and a treatment protocol is initiated, themethods of the invention may be repeated on a regular basis to evaluatewhether the methylation state of a gene or regulatory region thereof, inthe subject begins to approximate that which is observed in a normalsubject. Alternatively, or in addition thereto, the methods of theinvention may be repeated on a regular basis to evaluate whether thesymptoms associated with ovarian cancer have been decreased orameliorated. The results obtained from successive assays may be used toshow the efficacy of treatment over a period ranging from several daysto months to years. Accordingly, the invention is also directed tomethods for determining whether a subject is responsive to a particulartherapeutic regimen. The methods include determining the methylationstate of one or more genes or regulatory regions thereof, selected fromthe group consisting of GULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC,PAK3, MCAM, MGMT, KIFIA, CCNA1, SSRI, SSBP2, GSTP1, FKBP4, and VGF. Acomparison of the hypennethylation of the gene or regulatory regionthereof, as compared to that of a corresponding normal cell in thesubject or a subject not having the disorder is indicative of a subjectwho is responsive to the therapeutic regimen.

In one embodiment, the therapeutic regimen is administration of one ormore chemotherapeutic agent. In another embodiment, the therapeuticregimen is administration of one or more chemotherapeutic agents incombination with one or more demethylating agents.

Exemplary chemotherapeutic agents include, but are not limited to,antimetabolites, such as methotrexate, DNA cross-linking agents, such ascisplatin/carboplatin; alkylating agents, such as canbusil;topoisomerase I inhibitors such as dactinomicin; microtubule inhibitorssuch as taxol (paclitaxol), and the like. Other chemotherapeutic agentsinclude, for example, a vinca alkaloid, mitomycin-type antibiotic,bleomycin-type antibiotic, antifolate, colchicine, demecoline,etoposide, taxane, anthracycline antibiotic, doxorubicin, daunorubicin,carminomycin, epirubicin, idarubicin, mithoxanthrone,4-dimethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin,adriamycin-14-benzoate, adriamycin-14-octanoate,adriamycin-14-naphthaleneacetate, amsacrine, carmustine,cyclophosphamide, cytarabine, etoposide, lovastatin, melphalan,topetecan, oxalaplatin, chlorambucil, methtrexate, lomustine,thioguanine, asparaginase, vinblastine, vindesine, tamoxifen, ormechlorethamine. While not wanting to be limiting, therapeuticantibodies include antibodies directed against the HER2 protein, such astrastuzumab; antibodies directed against growth factors or growth factorreceptors, such as bevacizumab, which targets vascular endothelialgrowth factor, and OSI-774, which targets epidermal growth factor;antibodies targeting integrin receptors, such as Vitaxin (also known asMEDI-522), and the like. Classes of anticancer agents suitable for usein compositions and methods of the present invention include, but arenot limited to: 1) alkaloids, including, microtubule inhibitors (e.g.,Vincristine, Vinblastine, and Vindesine, etc.), microtubule stabilizers(e.g., Paclitaxel [Taxol], and Docetaxel, Taxotere, etc.), and chromatinfunction inhibitors, including, topoisomerase inhibitors, such as,epipodophyllotoxins (e.g., Etoposide [VP-16], and Teniposide [VM-26],etc.), and agents that target topoisomerase I (e.g., Camptothecin andIsirinotecan [CPT-11], etc.); 2) covalent DNA-binding agents [alkylatingagents], including, nitrogen mustards (e.g., Mechlorethamine,Chlorambucil, Cyclophosphamide, Ifosphamide, and Busulfan [Myleran],etc.), nitrosoureas (e.g., Carmustine, Lomustine, and Semustine, etc.),and other alkylating agents (e.g., Dacarbazine, Hydroxymethylmelamine,Thiotepa, and Mitocycin, etc.); 3) noncovalent DNA-binding agents[antitumor antibiotics], including, nucleic acid inhibitors (e.g.,Dactinomycin [Actinomycin D], etc.), anthracyclines (e.g., Daunorubicin[Daunomycin, and Cerubidine], Doxorubicin [Adriamycin], and Idarubicin[Idamycin], etc.), anthracenediones (e.g., anthracycline analogues, suchas, [Mitoxantrone], etc.), bleomycins (Blenoxane), etc., and plicamycin(Mithramycin), etc.; 4) antimetabolites, including, antifolates (e.g.,Methotrexate, Folex, and Mexate, etc.), purine antimetabolites (e.g.,6-Mercaptopurine [6-MP, Purinethol], 6-Thioguanine [6-TG], Azathioprine,Acyclovir, Ganciclovir, Chlorodeoxyadenosine, 2-Chlorodeoxyadenosine[CdA], and 2′-Deoxycoformycin [Pentostatin], etc.), pyrimidineantagonists (e.g., fluoropyrimidines [e.g., 5-fluorouracil (Adrucil),5-fluorodeoxyuridine (FdUrd) (Floxuridine)] etc.), and cytosinearabinosides (e.g., Cytosar [ara-C] and Fludarabine, etc.); 5) enzymes,including, L-asparaginase; 6) hormones, including, glucocorticoids, suchas, antiestrogens (e.g., Tamoxifen, etc.), nonsteroidal antiandrogens(e.g., Flutamide, etc.), and aromatase inhibitors (e.g., anastrozole[Arimidex], etc.); 7) platinum compounds (e.g., Cisplatin andCarboplatin, etc.); 8) monoclonal antibodies conjugated with anticancerdrugs, toxins, and/or radionuclides, etc.; 9) biological responsemodifiers (e.g., interferons [e.g., IFN-α, etc.] and interleukins [e.g.,IL-2, etc.], etc.); 10) adoptive immunotherapy; 11) hematopoietic growthfactors; 12) agents that induce tumor cell differentiation (e.g.,all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14)antisense therapy techniques; 15) tumor vaccines; 16) therapies directedagainst tumor metastases (e.g., Batimistat, etc.); and 17) inhibitors ofangiogenesis. Thus, in one embodiment, the therapeutic regimen is aadministration of cisplatin in combination with paclitaxel.

The materials for use in the methods of the invention are ideally suitedfor the preparation of a kit. As such, in another aspect, the inventionprovides a kit for detection of a methylated CpG-containing nucleic acidin determining the methylation status of one or more genes or regulatoryregions thereof. Such a kit may comprise a carrier device containing oneor more containers such as vials, tubes, and the like, each of thecontainers comprising one of the separate elements to be used in themethod. The kit may contain reagents, as described above fordifferentially modifying methylated and non-methylated cytosineresidues. One of the containers may include a probe which is or can bedetectably labeled. Such probe may be a nucleic acid sequence specificfor a promoter region associated with a gene selected from the groupconsisting of GULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM,MGMT, KIFIA, CCNA1, ESR1, SSBP2, GSTP1, FKBP4, and VGF. For example,oligonucleotide probes of the invention can be included in a kit andused for detecting the presence of hypermethylated nucleic acidsequences in a sample containing a nucleic acid sequence of the genesGULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA,CCNA1, ESR1, SSBP2, GSTP1, FKBP4, and VGF. The kit may also include acontainer comprising a reporter, such as an enzymatic, fluorescent, orradionucleotide label to identify the detectably labeled oligonucleotideprobe.

In certain embodiments, the kit utilizes nucleic acid amplification indetecting the target nucleic acid. In such embodiments, the kit willtypically contain both a forward and a reverse primer for each targetgene. Such oligonucleotide primers are based upon identification of theflanking regions contiguous with the target nucleotide sequence.Accordingly, the kit may contain primers useful to amplify and screen apromoter region of a gene selected from the group consisting of GULP1,CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA, CCNA1,ESR1, SSBP2, GSTP1, FKBP4, and VGF. If there is a sufficient region ofcomplementarity, e.g., 12, 15, 18, or 20 nucleotides, then the primermay also contain additional nucleotide residues that do not interferewith hybridization but may be useful for other manipulations. Forexample, such other residues may be sites for restriction endonucleasecleavage, for ligand binding or for factor binding or linkers orrepeats. The oligonucleotide primers may or may not be such that theyare specific for modified methylated residues. The kit may optionallycontain oligonucleotide probes. The probes may be specific for sequencescontaining modified methylated residues or for sequences containingnon-methylated residues. The kit may optionally contain reagents formodifying methylated cytosine residues. The kit may also containcomponents for performing amplification, such as a DNA polymerase anddeoxyribonucleotides. Means of detection may also be provided in thekit, including detectable labels on primers or probes. Kits may alsocontain reagents for detecting gene expression for one of the markers ofthe present invention. Such reagents may include probes, primers, orantibodies, for example. In the case of enzymes or ligands, substratesor binding partners may be used to assess the presence of the marker. Inparticular embodiments, the kit may include one or more primers orprimer pairs selected from the sequences set forth in SEQ ID NOs: 1-51.

The following examples are provided to further illustrate the advantagesand features of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

EXAMPLE 1

This example illustrates the comprehensive approach for identificationof ovarian cancer specific methylation markers.

Fourteen genes already known to be inactivated by promoter methylationin various cancers were evaluated in ovary tumors and normal samples byquantitative fluorogenic real-time methylation specific PCR (QMSP). Theselected genes were: GULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3,MCAM, MGMT, KIFIA, CCNA1, ESR1, SSBP2, GSTP1, FKBP4, and VGF.

An evaluation set consisting of 33 ovarian tumor samples and threenormal ovarian cell lines were first tested (see Table 1). Based onsensitivity and specificity, five genes (PGP9.5, HIC1, MCAM, ESR1, andVGF) were selected for further analysis in an independent test set of 24samples (see Tables 2 and 3). In this set of samples, MCAM and HIC1methylation was observed in 96% (23/24) and 75% of the (18/24) samples,respectively. ESR1 and PGP9.5 promoter methylation was found in 42%(10/24) samples, and 33% (8/24) of the tumor samples, respectively. Thehigh frequencies of hypermethylation found by QMSP for these five genesin two independent sets of tumor samples suggest that these epigeneticalterations are important in ovarian cancer development.

TABLE 1 Demographic and clinical characteristics of ovarian cancerpatients No. (%) patients characteristics Evaluation set Independenttest set Age Median (range) 57 (23-79) 56 (37-77) ≧60 13 (39) 9 (38) <6020 (61) 15 (62) Race Caucasian 24 (73) 24 (100) African-american 8 (24)0 (0) Unknown 1 (3) 0 (0) Stage Early I 15 (46) 6 (25) II 1 (3) 4 (17)Advanced III 14 (42) 12 (50) IV 3 (9) 1 (4) Unknown 0 (0) 1 (4) GradeBorderline 12 (36) 0 (0) GX 0 (0) 1 (4) G1 0 (0) 2 (8) G2 8 (24) 5 (21)G3 13 (40) 12 (50) Unknown 0 (0) 4 (17) Tumor type EOC 33 (100) 18 (75)Germ cell 0 (0) 1 (4) Stromal 0 (0) 0 (0) Secondary (mets.) 0 (0) 4 (17)Unknown 0 (0) 1 (4) EOC Histology Serous-papillary 33 (100) 11 (46)Endometrioid 0 (0) 4 (17) Mucinous 0 (0) 2 (8) Squamous 0 (0) 0 (0)Undifferentiated 0 (0) 1 (4) Unknown 0 (0) 6 (25) Chemotherapy ResponseYes 0 (0) 13 (54) No 0 (0) 10 (42) Unknown 33 (100) 1 (4) Smoking statusNon-smoker 16 (48) 19 (79) Smoker 2 (6) 5 (21) Unknown 15 (46) 0 (0)Alcohol consumption Current 6 (48) 2 (8) No 12 (6) 22 (92) Unknown 15(46) 0 (0) Chemotherapy Yes 11 (33) 23 (96) No 9 (27) 1 (4) Unknown 13(40) 0 (0) Recurrence Yes 4 (12) 14 (58) No 0 (0) 10 (42) Unknown 29(88) 0 (0) Metastasis Yes 1 (3) 0 (0) No 32 (97) 0 (0) Unknown 0 (0) 24(100) Deaths Yes 9 (27) 11 (46) No 24 (73) 13 (54) Unknown 0 (0) 0 (0)Total 33 (100) 24 (100)

An established pharmacologic unmasking strategy that they performedusing three human ovarian cancer cell lines and three normal ovariancell lines. Computational analysis was then used to identifycancer-specific methylated genes. 43 highly-ranked genes were tested,ten novel genes (DKK1, CSGALNACT2, CAV1, CLIP4, TFGB2, GATA6i1, BAMB1,DNAJC6, NT5E, and GULP1) were identified as potential cancer-specificmethylated genes.

TABLE 2 Promoter methylation frequency for 14 genes analyzed in theevaluation set of samples and 5 genes in the independent test setIndependent Set Evaluation Set Methylation Methylation Positive %Positive % Sensitivity Specificity Gene Tumor Controls P¹ Tumor ControlsP¹ P² (%) (%) ESR1 30 (10/33) 0 (0/9) 0.086 42 (10/24) 50 (3/6) 1 0.36735 75 MCAM 45 (15/33) 0 (0/9) 0.016 96 (23/24) 50 (3/3) 0.018 0.002 66.775 HIC1 67 (22/33) 0 (0/9) <0.0001 75 (18/24) 33 (2/6) 0.061 0.0004 70.283.3 PGP9.5 21 (7/33)  0 (0/9) 0.314 33 (8/24)   0 (0/6) 0.155 0.03126.3 100 VGF 24 (8/33)  0 (0/9) 0.118 54 (13/24)  0 (0/6) 0.024 0.01636.8 100 CCNA1 3 (1/33) 0 (0/9) ND³ ND PAK3 18 (6/33)  100 (9/9)  ND NDSSBP2 9 (3/33) 11 (1/8)  ND ND APC 15 (5/33)  0 (0/9) 0.567 ND FKBP4 9(3/33) 0 (0/9) 0.577 ND GSTP1 0 (0/33) 0 (0/9) ND ND KIF1A 6 (2/33) 11(1/9)  ND ND MGMT 3 (1/33) 0 (0/9) ND ND AIM1 3 (1/33) 0 (0/9) ND ND¹Fisher's exact test (two sided); ²Fisher's exact test (two sided) forcombined group (evaluation and independent); ³ND = Not analyzed

TABLE 3 Spearman correlation matrix of promoter methylation of 5 genesin ovarian tumor samples Genes ESR1 MCAM HIC1 PGP9.5 VGF ESR1 1.00 MCAM0.24 1.00 HIC1 0.30 0.67* 1.00 PGP9.5 0.23 0.30 0.09 1.00 VGF 0.23 0.48*0.47* 0.31 1.00

EXAMPLE 2

This example is intended to identify ovarian cancer methylation specificpatterns by applying a pharmacologic unmasking method. This approachconsists of performing expression microarray on 15 ovarian tumorsamples, 10 ovarian surface epithelium brushing (normal ovary), 3ovarian cancer derived cell lines (A2780, 2008, IGROV) and 3 normal celllines (derived from ovarian inclusion cysts: OSE2A, OSE2B, OSE7). Theovarian cancer cell lines were paired as cisplatin resistant vs.cisplatin sensitive, as follows: 2008C13 vs. 2008; A2780CP vs. A2780;and IGROVCP vs. IGROV. For each cell line, a control (untreated) and acell treated with 5AZA (re-expression experiment) were also tested. Allcells have been treated with a global demethylating agent which willcomprehensively uncover genes silenced by promoter hypermethylation. Theinvestigation searched for genes that are differentially expressedbetween normal cell lines/tissue samples and tumor cell lines/tissuesamples, being down-regulated in cancer samples/cell lines when comparedto normal, and finally that have been re-activated after the treatmentwith the demethylating agent.

The microarray platform used was U133 Plus 2.0 from Affymetrix (SantaClara, Calif.), which contains over 47,000 transcripts. Applying thecriteria mentioned above, a list with 250 transcripts (array probes)that corresponded to 88 genes was obtained. Genes having full lengthtranscripts were first selected, which left 79 genes. Any genes withoutCpG islands in their promoters were then excluded, resulting in a totalof 67 genes. Two of these genes have been extensively reported as beinghypermethylated in cancer (DAPK and APC), so they were also excluded.Primers for 43 of the 65 genes were then successfully designed to screenthe CpG islands contained in the promoter region and determine theirmethylation status by bisulfite sequencing in 3 tumor cell lines and 3normal cell lines. Ten candidates where then obtained. And work beganwith two of them (GULP1 and CSGALNACT2).

EXAMPLE 3

Briefly, cells were split to low density (1×10⁶ cells/T-75 flask) 24hours before treatment. Stock solution of 5Aza-dC (Sigma, St. Louis,Mo.) was dissolved in DMSO (Sigma). Cells were treated with 5 μM5-Aza-deoxycytidine for 5 days. The medium was changed every 24 hours.Baseline expression was established by mock-treated cells with the samevolume of DMSO or ethanol.

Oligonucleotide microarray analysis and QRT-PCR analysis. Total cellularRNA was isolated using the RNEASY RNA isolation kit (Qiagen, Valencia,Calif.) according to the manufacturer's instructions. Oligonucleotidemicroarray analysis was carried out using the GENECHIP U133plus2Affymetrix expression microarray (Affymetrix, Santa Clara, Calif.).Samples were converted to labeled, fragmented, cRNA per themanufacturer's protocol for use on the expression microarray. Signalintensity and statistical significance were established for eachtranscript using dChip version 2005. 2-fold increase based on the 90%confidence interval of the result and expression minus baseline >50 wasused as the statistical cutoff value after 5Aza-dC to identify silencedcandidate genes.

A sorting method, as described below. The U133A microarray platform(Affymetrix, Santa Clara Calif.) has approximately 14,500 probe sets.This resulted in 250 genes deemed significant. The top 88 of thesetargets were comprehensively evaluated. Presence of CpG islands in thesegenes was determined by MethPrimer. In order to not exclude genesoutside the U133A platform, also considered were all other genes in theU133plus2 platform on the sole basis of 5-aza upfold regulation. 43genes were studied that had an experimental versus baseline expression(E/B)>2.0, based on the 90% confidence interval and E-B>50. All geneswere then studied for the presence of CpG islands in promoters or thefirst intron. Initially, an in silico approach was used to confirm thepresence of a CpG island using the UCSC genome browser which relies onGC content of>50%, >200 bp, >0.6 observed to expected CG's.

DNA extraction. Samples were centrifuged and digested in a solution ofdetergent (sodium dodecylsulfate) and proteinase K, for removal ofproteins bound to the DNA. Samples were first purified and desalted withphenol/chloroform extraction. Digested sample was subjected twice toethanol precipitation, and subsequently resuspended in 500 μL of LoTE(EDTA 2.5 mmol/L and Tris-HCI 10 mmol/L) and stored at −80° C.

Bisulfite treatment. DNA from the tissue samples was subjected tobisulfite treatment, as described previously (Herman et al., Proc NatlAcad Sci USA 1996;93:9821-6). In short, 2 μg of genomic DNA wasdenatured in 0.2 M of NaOH for 30 minutes at 50° C. The denatured DNAwas then diluted into 500 μL, of a solution of 10 mmol/L hydroquinoneand 3 M sodium bisulfite and incubated for 3 hours at 70° C. Afterincubation, the DNA sample was purified with a SEPHAROSE column (WIZARDDNA Clean-Up System; Promega, Madison, Wis.). Eluted DNA was treatedwith 0.3 M of NaOH for 10 minutes at room temperature, and precipitatedwith ethanol. This bisulfite-modified DNA was subsequently resuspendedin 120 μL of LoTE (EDTA 2.5 mmol/L and Tris-HC1 10 mmol/L) and stored at−80° C.

Bisulfite sequencing. Bisulfite sequence analysis was performed to checkthe methylation status in primary tumors and normal tissues, as well asin cell lines (Tables 5A and 5B). Bisulfite-treated DNA was amplifiedusing primers designed using the MethPrimer program (Li and Dahiya,Bioinformatics 18(11):1427-31, 2002) to span areas of CpG islands in thepromoter or first intron. (see Tables 4A-4C below for primer sequences;ACTB =βactin, which is used as a housekeeping gene to which allmethylation values have been normalized). The PCR products weregel-purified using the QIAQUICK gel extraction kit (Qiagen), accordingto the manufacturer's instructions. Each amplified DNA sample wasapplied with nested primers to the Applied Biosystems 3700 DNA analyzerusing BD terminator dye (Applied Biosystems, Foster City, Calif.).

TABLE 4A QMSP Forward Primers Gene Forward 5′-3′ (primer) SEQ ID NO:ACTB TGGTGATGGAGGAGGTTTAGTAAGT  1 AIM1 CGCGGGTATTGGATGTTAGT  2 APCGAACCAAAACGCTCCCCAT  3 CCNA1 TCGCGGCGAGTTTATTCG  4 ESRGGCGTTCGTTTTGGGATTG  5 GSTP1 AGTTGCGCGGCGATTTC  6 HIC1GTTAGGCGGTTAGGGCGTC  7 KIF1A GCGCGATAAATTAGTTGGCGATT  8 MCAMAGAATTTAGGTCGGTTTTTATCG  9 MGMT CGAATATACTAAAACAACCCGCG 10 PAK3TTACGGTCGTCGTTATTATCG 11 PGP9.5 CGGCGAGTGAGATTGTAAGGTT 12 SSBP2ATTTTTGCGGTCGTAGCGGT 13 VGF GGATAGCGTTCGTAGGCG 14 FKBP4GTTCGTGGTGACGGTCGGTTTCGGG 15 GULP1 TGACGTTTGTTATGGTAGCG 16 CSGALNACT2TTAGTTGAGGGTCGTGGTCG 17

TABLE 4B QMSP Probes SEQ Gene Probe 5′-3′ (6-FAM-5′-3′-6-TAMRA) ID NO:ACTB ACCACCACCCAACACACAATAACAAACACA 18 AIM1 GGGAGCGTTGCGGATTATTCGTAG 19APC CCCGTCGAAAACCCGCCGATTA 20 CCNA1 CGTTATGGCGATGCGGTTTCGG 21 ESRCGATAAAACCGAACGACCCGACGA 22 GSTP1 CGGTCGACGTTCGGGGTGTAGCG 23 HIC1CAACATCGTCTACCCAACACACTCTCCTACG 24 KIF1A CCTCCCGAAACGCTAATTAACTACGCG 25MCAM ACAATATCAAACCGACGACAACGAC 26 MGMT AATCCTCGCGATACGCACCGTTTACG 27PAK3 AACCAAAAAAAATAAAAAATCACAACCG 28 PGP9.5 TTCGGTCGTATTATTTCGCGTTGCGTAC29 SSBP2 ATATCCAAAACGCCGCGAAACTCC 30 VGF GCGCCCAAAAACGACGTAAACCTAAATAC31 FKBP4 CAAACTACGAAATAACAATAACGACGC 32 GULP1 CGGCGGGGGGTCGGTGAGTA 33CSGALNACT2 CGAACGCTACCTAAACCCCCGAA 34

TABLE 4C QMSP Reverse Primers Gene Reverse 5′-3′ (primer) SEQ ID NO:ACTB AACCAATAAAACCTACTCCTCCCTTAA 35 AIM1 CCGACCCACCTATACGAAAA 36 APCTTATATGTCGGTTACGTGCGTTTATAT 37 CCNA1 CCGACCGCGACAAACG 38 ESRGCCGACACGCGAACTCTAA 39 GSTP1 GCCCCAATACTAAATCACGACG 40 HIC1CCGGGCGCCTCCATCGTGT 41 KIF1A CTCGACGACTACTCTACGCTAT 42 MCAMACGCAAAATTCTTCTCCCAAAA 43 MGMT GTATTTTTTCGGGAGCGAGGC 44 PAK3ACCGAAAATTCTACCCTTCG 45 PGP9.5 GAACGATCGCGACCAAATAAATAC 46 SSBP2TTCTACGACAAATCTAACGAA 47 VGF AAAAACCGAATTCCCCACCCCG 48 FKBP4ATCCGCTACGCCTACGACG 49 GULP1 CGGTGGGGAAATCGTGGA 50 CSGALNACT2CGCGTATTTGTTAGACGTGCG 51

TABLE 5 Methylation status of 5 candidate genes in ovarian cellsanalyzed by bisulfite sequencing GENE OSE2A OSE2B OSE7 IGROV IGROVCPA2780 A2780CP 2008 2008C13 CANCER SPECIFIC RELATED GENES T2 U U U M M UU U U C4 U M U M M M M U U BI X X U M M X U M M G1 U U U M M U U M M N5EU U U M M U U U U DRUG RESPONSE RELATED GENES D1 M M X M M M U U M D5 MM U M M U M M M G6 U U U U M U U U U A2 M M U U/M M U M M M C1 U U M M UM U U U U Unmethylated M Methylated X No result

As shown in Table 5, D1=DKK1, A2=CSGALNACT2, C1=CAV1, C4 =CLIP4,T2=TFGB2, G6=GATA6i1, B1=BAMB1, D6=DNAJC6,NT5E=NT5E, and G1=GULP1.

Quantitative methylation-specific PCR (QMSP). To selectively amplifyhypermethylated promoter regions in genes of interest, probe and primerswere designed using data from bisulfite sequencing of primary tumorswhich are complimentary only to bisulfite-converted sequences known tobe methylated in tumor (see Tables 4A-4C). Primer combinations werevalidated using in vitro methylated and demethylated controls.

qRT-PCR. Total RNA was measured and adjusted to the same amount for eachcell line, and then cDNA synthesis was performed using oligo-dt with theSUPERSCRIPT first-strand DNA synthesis kit (Invitrogen). The final cDNAproducts were used as the templates for subsequent PCR with primersdesigned specifically for each candidate gene. GAPDH was examined toensure accurate relative quantitation in qRT-PCR. qRT-PCR heat maps weregenerated by median-normalization by gene, logged and heat mapsgenerated using Excel.

Fifteen ovarian tumor samples, ten normal ovarian surface epitheliumbrushing samples, three normal ovarian cell lines, and three cancer celllines (DAC treated and mock treated) were assayed for mRNA expression onthe Affymetrix U133A mRNA expression microarray platform (16,383 probesets) compiled from prior work and public sources of expression(oncomine.org). In order to select the best methylation biomarkers inovarian cancer, all these criteria are combined using a score scheme, asfollows:

-   -   down-regulation in cancer vs. normals: the results from the        RankProd FDR analysis are used        -   results are ranked, based on p-value; the score given to a            probe reflects this ranking: Ranking            Score=tan_(1-rank/54675)        -   the FDR itself is taken into account: FDR Score=tan ƒ(1-FDR)        -   the fold change (FC) A is scored next, Δ_(max) being the            maximal FC observed: FC Score=tan ƒ(ΔΔ_(max)); in other            words, the tangens function of the ratio between delta and            delta max.        -   the tangens function is chosen as all values are between 0            and 1, and the tangens between these values range from 0 to            1.56 in a non-linear way, with somewhat (but not too            extreme) more increase in score for values approaching 1            (being the best possible). This way, probes with a very good            profile have a higher chance to acquire a high score and to            be selected.    -   downregulation in cancer cell lines vs. normal cell lines and        cancer cell lines vs. DAC-treated cancer cell lines is examined        the same way    -   low expression in tumor samples and cancer cell lines is        examined as follows:    -   for every sample, the maximal expression is determined; for each        probe its expression level is compared to the maximal expression        level in that sample: expression score=1        −_(probe expressionmaximal expression sample)    -   for all samples, the sum is calculated.

Using this score scheme, 11 values are generated (3 for down-regulationin tumors vs. normal; one for expression in tumor samples; 3 fordown-regulation in cancer cell lines vs. normal cell line; one for lowexpression in cancer cell lines and three for upregulation after DACtreatment). For each of these values, the percentile of the probe iscalculated: for each probe, for a specific score it is calculated whereit is ranked, expressed in percentages versus the scores of all probes.For instance a probe with a score of 95% of a particular probe wassituated at the top 5% probes with best scores. Next, we determine thenumber of scores where a probe was at least in the best 5% as well asthe average percentile of all scores for this probe. The probes aresorted primary on the number of scores in the best 5% percentile,followed by the average percentile score. The sorting methodology allowsto select a top-selection without having to choose thresholds orconditions. We chose to analyze the top 250 probes.

EXAMPLE 4

This example demonstrates how identification of tumor suppressor genes(TSGs) silenced by CpG methylation uncovers the molecular mechanism oftumorigenesis and potential tumor biomarkers.

Performing a pharmacologic unmasking technique, it was observed thatGULP1, a molecule not previously related to cancer, is down-regulated inovarian tumor tissues when compared to normal ovary cells. GULP1 is acytoplasmic adaptor protein with a phosphotyrosine binding domain thatplays a role in one of two partially redundant pathways that lead to theengulfment and clearance of apoptotic cells, according to severalgenetic studies performed in Caenorhabditis elegans.CED-2/CrkII,CED-5/Dock180, and CED-12/ELMO1 act together in one pathwaythat finalizes in the regulation of CED-10, which promotes cytoskeletalreorganization during engulfment of apoptotic cells. CED-1/MEGF10,CED-7/ABCA1, and CED-6/GULP collaborate together in the other pathway(11, 17-19), which at the end converge with the first pathway at CED-10to mediate actin rearrangement and the subsequent engulfment.

An expression microarray was performed on 15 ovarian tumor samples, 10ovarian normal surface epithelium, 3 ovarian cancer cell lines and 3normal cell lines. All cells were treated with a demethylating agentthat comprehensively uncovers genes silenced by promoterhypermethylation. The selection of the best candidates was based uponthe differential expression between normal and tumor samples (or celllines), them being downregulated in cancer when compared to normal, andfinally the re-activation after the treatment with the demethylatingagent.

One of the promising genes was GULP1, which expression pattern was thenassessed by Reverse Transcriptase PCR and Western Blot and compared toits methylation status in the same six ovarian cell lines and showedcorrelation between absence of expression and presence of methylation(or the opposite situation).

Quantitative fluorogenic methylation specific PCR (QMSP) primers werethen developed for GULP1. 437 ovarian tumor samples, 17 borderlinetumors, 19 cystadenoma samples and 13 normal ovarian samples wereprofiled, finding 34.7% (151/437), 11.7% (2/17), 10.5% (2/19) and 0%(0/13) of methylation frequency, respectively (establishing an empiriccutoff). Using Fisher's exact test, a significant increase inmethylation was observed when comparing tumors with cystadenomas andnormals (p=0.0439 and p=0.0131, respectively). Late stage tumors alsoshowed a higher frequency of methylation versus early stage (p=0.004).

TABLE 6 Demographic and clinical characteristics of ovarian cancerpatients Characteristics No. (%) patients Age Median (range) 61 (22-90)Stage Early 96 (25%) Advanced 288 (75%) Chemotherapy Response Yes 138(35.9%) No 53 (13.8%) Unknown/Not evaluable 193 (50.3%) EOC HistologySerous-papillary 238 (62%) Endometrioid 38 (9.9%) Mucinous 43 (11.2%)Clear Cell 18 (4.7%) Adeno 14 (3.6%) Other 32 (8.3%) Unknown 1 (0.3%)Deaths Yes (by the disease) 208 (54.2%) No 161 (41.9%) Unknown 15 (3.9%)Samples Tumor 384  Borderline 17 Cystadenoma 19 Normal 16

So far, reduced log growth rates have been demonstrated aftertransiently overexpressing GULP1 in one ovarian cell line (IGROVCP).These preliminary data indicate that GULP1 may be an ovarian cancerbiomarker.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A kit useful for the detection of a methylatedCpG-containing nucleic acid in determining the methylation status of oneor more genes or regulatory regions thereof, selected from the groupconsisting of GULP1, CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM,MGMT, KIFIA, CCNA1, ESR1, SSBP2, GSTP1, FKBP4, VGF, and any combinationthereof, comprising a carrier element containing one or more containerscomprising a first container containing a reagent that modifiesunmethylated cytosine and a second container containing primers foramplification of the one or more genes or regulatory regions thereof,wherein the primers distinguish between modified methylated andunmethylated nucleic acid.
 2. The kit of claim 1, wherein the modifyingregent is bisulfite.
 3. The kit of claim 1, wherein the second containercontains primers for amplification of two or more genes or regulatoryregions thereof.
 4. The kit of claim 1, further comprising a panel oftwo or more genes selected from the group consisting of GULP1,CSGALNACT2, PGP9.5, HIC1, AIM1, APC, PAK3, MCAM, MGMT, KIFIA CCNA1,ESR1, SSBP2, GSTP1, FKBP4, VGF, and any combination thereof.